Project Summary: Mesenchymal-to-epithelial transitions (METs) play fundamental roles in many tissue-shaping processes including embryonic development, fibrosis, and stem cell reprogramming, yet we know little about the biomechanical cues that initiate and regulate METs, what pathways are regulated by METs, and how newly epithelialized clusters grow in size. In this project we seek to define the biophysical and biomechanical principles that guide MET in vivo during early development of the heart and ex vivo within 3D mesenchymal aggregates. During heart development, in vivo METs occur as bilateral populations of heart progenitor cells migrate toward the ventral midline. We have developed one experimental model system in which METs shape the early heart primordia in Xenopus laevis and another where METs occur spontaneously in ex vivo 3D aggregates of X. laevis embryonic mesenchymal cells. These models are compatible with live microscopy and are uniquely accessible to combined biophysical, biomechanical, cell biological, and genetic analysis. Using these models, studies in our lab have revealed a complex interplay between cell and tissue biomechanics and MET both in vivo and ex vivo. Intra vital imaging of heart precursor cells reveals that they change their mechanical mode of migration as they undergo MET. Our lab has also found that METs in both models are highly sensitive to tissue tension and cell contractility. In our first aim we focus on describing phenotypic changes in heart progenitor cells as they undergo MET and how MET alters their mechanics and migration through a cell dense microenvironment. Our second aim seeks to identify mechanical and molecular pathways that drive MET in 3D mesenchymal aggregates, focusing on pathways that transduce mechanical cues in this process. In our third aim, we explore the cell biology of MET initiation and spreading in 3D aggregates and test our findings within a 3D ex vivo model of heart formation. We focus our studies on in vivo and ex vivo models of MET to identify and test the role of mechanical cues in MET during heart formation and how biomechanical and biophysical processes integrate with the cell biological processes that drive tissue assembly. Due to the similarities of developmental METs with METs in other systems, our findings will likely expose common pathways regulating METs during regeneration, wound healing, fibrosis, and cancers metastases.